Asporogenic protease-producing strains of bacillus subtilis

ABSTRACT

A new mutant strain of Bacillus subtilis which has the property of being substantially asporogenic while showing proteolytic activity similar to that of the native strain from which it was derived. The process for preparing the new mutant strain is based on the discovery that the non-sporulated cells of Bacillus subtilis are not resistant to chloroform vapor whereas the spores are resistant.

United States Patent Aubert et al.

[451 Sept; 3, 1974 ASPOROGENIC PROTEASE-PRODUCING STRAINS OF BACILLUSSUBTILIS Inventors: Jean-Paul Aubert, Montrouge;

Robert Longin, Meudon La Foret; Jacqueline Millet, Paris, all of FranceAgence Nationale de Valorisation de la Recherche (Anvar), Paris, FranceFiled: July 25, 1972 Appl. No.: 275,069

Assignee:

Foreign Application Priority Data July 28, 1971 France 71.27671 US. Cl.195/65, 195/78, l95/103.5 R,

195/96 Int. Cl. ClZk l/00, Cl2d 13/10 Field of Search 195/65, 66 R, 96

[56] References Cited UNITED STATES PATENTS 3,740,318 6/1973 Churchillet al. 195/65 FOREIGN PATENTS OR APPLICATIONS 2,018,451 10/1970 Germany195/66 R Primary Examiner-Lionel M. Shapiro Attorney, Agent, orFirm-Edward J. Brenner 57 ABSTRACT 3 Claims, No Drawings ASPOROGENICPROTEASE-PRODUCING STRAINS OF BACILLUS SUBTILIS This invention relatesto the production of enzymes from strains of Bacillus subtilis.

It is known that the proteolytic enzymes obtained by fermenting strainsof Bacillus subtilis are widely used in industry, especially in thedetergent industry.

However, workers exposed to the commercial-grade dust of proteolyticenzymes have been found to suffer from allergic reactions (asthma,coughing, chest pains). Similarly, certain workers have been found tosuffer from allergies affecting the skin.

Although this has not been demonstrated with any degree of certainty, itis generally thought that these allergic reactions are caused by thepresence of spores in commercial-grade enzymes. The standardsestablished by common agreement between enzyme manufacturers andmanufacturers of domestic detergents limit the number of spores per gramof commercial enzymes although, hitherto, these standards have beendifficult to attain.

According to one of its aspects, the invention relates to a new mutantstrain of Bacillus subtilis which has the property of beingsubstantially asporogenic whilst showing proteolytic activity similar tothat of the native strain from which it is derived.

The invention also relates to a process for mutating a native strain ofBacillus subtilis and isolating the asporogenic strain according to theinvention.

The mutant strain obtained by this process has the same identificationcharacteristics as the original native strain except that it; does notsporulate. Its identification which is thus already possible bycomparison with the well-known and defined native strain is facilitatedby the fact that the mutant strain has been deposited in the collectionof the Pasteur Institute in Paris and given the identification number71-l00l.

By virtue of the fact that the mutant strain according to the inventionis no different from the native strain already used in the production ofprotease, i.e., an extracellular enzyme used in detergents, apart fromits asporogenic character, this mutant can readily be subjected to knownprocessesfor commercially growing the native strain with a view toobtaining enzymes suitable for use in household detergents.

In addition to the fact that they do not give rise to any of theallergic reactions referred to above, the enzymes thus obtained byapplying a conventional process to the new mutant strain according tothe invention also have the advantage of not giving off the unpleasantodour attending the use of detergents containing enzymes obtained fromspore-forming strains. Finally, the absence of spores avoids anysubsequent microbial growth.

Preparation of the mutant strain according to the invention is based onthe following observation:

We have found that the non-sporulated cells of Bacillus subtilis are notresistant to chloroform vapour whereas the spores are resistant. It isthus possible to mark the colonies of asporogenic strains (sp) becausethese'colonies are not replicated after exposure to chloroform vapours.

Accordingly, the process comprises (A) subjecting the spores of thenative strain to a mutagenic treatment, (B) spreading the sporessubjected to the mutagenic treatment over an agar-containingnutrientbroth accommodated in Petri dishes to obtain after incubationspore-containing colonies and (C) isolating colonies of the asporogenicstrain by comparison between the homologous Petri dishes of one seriesemanating from operation (B) and having been subjected to treatment withchloroform vapours followed by replication and incubation, and a serieshaving undergone the same operations except for exposure to thechloroform vapours, and finally (D) selecting the mutant strainsatisfying the required sporulation conditions from the mutant strainsisolated showing proteolytic activity.

In one embodiment of this process, the mutagenic treatment (A) of thespores of the native strain of Bacillus subtilis comprises initiallyheating a suspension of spores for 10 minutes to C., followed bygermination in Difco nutrient broth in the presence of chloramphenicolug/ml), the culture being stirred for 2 hours at 39C., and finally bythe actual mutagenic treatment with N-methyl-N'-nitro-N-nitrosoguanidine(50 tog/ml), the culture being stirred for 1 hour at 37C. The culturemedium used for germination has the following composition:

for 1 litre of distilled water NaOH quantity sufficient to adjust pH to6.8 7.0

A dilute form of the suspension of spores germinated same composition asthat used for germination, but additionally contains 20 g/litre of agar.After incubation for 48 hours at 37C., the sp colonies contain spores.

Isolation (C) which includes a treatment with chloroform vapours appliedto certain colonies comprises the following operations:

a. colonies of series I are replicated in other Petri dishes containingthe same medium (series II);

b. colonies of series I are then exposed to chloroform vapours for 45minutes at the temperature of the laboratory, after which the coloniesthus treated are replicated in a new series of Petri dishes (seriesIII);

c. series II and III are incubated for 24 hours at 37C.;

d. the homologous dishes of series II and III are compared.

The sp colonies are absent from series lIl but present in series Ilbecause the cells are killed by the chloroform vapours. Samples of thesp colonies are taken from series II and then isolated again. After thesecond isolation, the strain retained for their sp character are testedfor their proteolytic activity. To this end, the medium usedis'preferably agar-containing Difco nutrient broth with casein added.For example, a Difco medium of the following composition is used:

for 1 litre of distilled water Difco nutrient broth 16 g MgSO '7H,O 0.50g KCl 2.0 g MnCl,4H O l' M 2 ml FeSO -7H,O l0M 2 ml CaCl -2H,O 1.5% 20ml NaOH quantity sufficient to adjust pH to 6.8 7.0 agar 20 S The mediumis mixed in equal volumeswith a solution containing 20 g of casein perlitre of distilled water, the pH-value being adjusted to 6.8 7.0

The colonies showing proteolytic activity (Pr are surrounded by aproteolysis ring.

Selection (D) of the mutant strain is made as follows:

Out of 35,000 colonies examined in series 1, seven mutant strains wereisolated. They were studied in liquid Difco nutrient broth with the samecomposition as the broth previously used for germinating the spores inorder accurately to determine their sporulation rate fer. After themixture has been incubated for 30 minutes in. a water bath at 37C., thenon-hydrolysed azocasein is precipitated with 2 ml of 10%trichloroacetic acid. The mixture is filtered. 1.5 ml of 0.5 N NaOH areadded to l .5 ml of the filtrate, followed by a reading at 440 nm. Oneunit (UA) corresponds to 1 mg of azocasein hydrolysed for 30 minutes at37C. under the conditions specified above. I

The results are set out in Table l below:

strain y proteolytic sporulation 4 activity sp/ml.

UA/ml B native 14 8.10 B l2 l4 0 B 22 l 0 B 6| 9 r 5.10 Mutants B 8216.3 1.4 l0

B I ll 2.8 0 B I 12 l l 4.4 l0 B143 14.5 l.2' 10 Labile rod (width 0.8to l.0 ,u; length 2.4 to 2.6 t).

In the exponential growth phase, the cells are in the form of more orless long chains (4 to 10 cells). After growth, the chains are shorter(2 to 4 cells) and the cells are less long.

Gram-positive colouration.

The spores formed are oval (0.8 1.4/1.1 11.).

Mutant strain ldentical, but does not form spores.

, 2. Appearance of colonies on the solid agarcontaining Difco nutrientbroth having the composition specified for treatment (B) with themutagenic agent.

Native strain:

Round, white, dull smooth-edged colonies flattened at the centre andopaque. After 48 hours at 37C., a yellow pigment is excreted.

Mutant strain Identical colonies except that, after 3 to 4 days at 37C.,the colonies become translucent (destruction of the cells). Yellowpigment is again excreted.

b. Physiology Growth in Difco nutrient broth having the same compositionas the media used for the germination operations (A), for examining themutant strains in phase (D) and for examining the morphology of thecells (a) above.

Native strain Mutant strain Growth rate at 30C #1 0.49 t, 0.49(division/hour) 0.32 0.32 Number of cells (maximum/ml) 9.10 9.!0Proteolytic activity in UA/ml -14 -14 Number of spores/ml 810 0Prototrophy c. Comparison of the properties of the enzymes excreted bythe nativestrain and the mutant strain 1. Heat resistance.

a. Having reached maximum proteolytic activity, the cells of one cultureare eliminated by centrifuging. Aliquot portions of the supernatantphase are heated in a water bath for 10 minutes at differenttemperatures (40, 45, 50, 55 75C). The residual proteolytic activity ismeasured by the process described above. The percentage ofresidualproteolytic activity is recorded on a graph as a function of temperaturefor the two types of enzyme. It is found that, in both cases, the,

temperature at which 50 percent of the original activity log A/Ao kt loge for a temperature of C. The gradient of each straight line [k log e]makes it possible to determine the velocity constant of thedenaturation' reaction (k). If more than one straight line is obtained,

it means that there is more than one enzyme in the supernatant phase.The gradient of each straight line is characteristic of an enzyme.

The gradients observed for the enzymes of the mutant and of the nativestrain suggest that the supernatant phases contain twoproteolyticenzymes of which one shows hardly any resistance to heat.

So far as the most resistant enzymes are concerned, the gradients k loge are the same for the enzyme emanating from the mutant and for theenzyme emanating from the native strain. For these enzymes, the velocityconstant of the denaturation reaction is:

k (70C) 0.1 min are not inhibited by o-phenantroline hydrochloride b.The degree of inhibition caused by diisopropyl fluorophosphate IO M)is'of the order of 95 percent for the mutant and the native strain.Accordingly, the principal enzyme would be seryl-protease in both cases.

Overall, the foregoing examination shows that the mutant strainaccording to the invention is an asporogenie mutant of the native strainof Bacillus subtilis which does not have any effect either uponproduction or upon the properties of the extracellular protease, i.e.,the enzyme produced by fermentation from the strain, except in regard tothe formation of spores.

The enzyme produced by fermentation of the Bacillus subtilis strain ispresent in solution in the aqueous phase of the culture broth. Thefollowing procedure can be adopted for producing the enzymes accordingto the invention on a commercial scale:

A fermentation process is carried out in a variablesized fermenteraerated in depth, agitated and equipped with a heat-regulating system.The culture medium contains organic nitrogen compounds such asdistillery residues, maize maceration liquor, soya flour, casein,mineral elements etc. This culture medium is inoculated with anasporogenic Bacillus subtilis culture (strain according to the inventionas deposited) obtained in agitated flasks or from an inoculum fermenter.i

The aeration required amounts for example to between 100 and 500 m" perhour for a fermenter with a useful capacity of m.

Fermentation is carried out at 37C. However, the fermentationtemperature can be in a range from 30 to 38C. The pH-value is adjustedto 7.0 at the beginning of fermentation.

After having been inoculated, the culture medium is sterilised with livesteam for 30 minutes at 121C.

The air injected at the bottom of the fermenter is sterilised by passagethrough a cylinder filled with glass wool.

On completion of fermentation, the liquid phase is separated from thebacteria and solid particles by fermentation, generally in a filterpress comprising :1 Nylon cloth. A filtration additive is added to thefermentation juice to be filtered.

The filtrate is precipitated with a solvent such as ethanol, butanol, oreven with ammonium sulphate. The precipitate obtained is centrifuged ina centrifuge, for example of the Sherpless type and filtered in a filterpress. The wet cake obtained is introduced in trays into an oven inwhich it is dried in vacuo at a temperature not exceeding 60C.

The powder obtained does not contain any spores. It can be used indetergents made up in accordance with the usual formulations and-doesnot produce any of the allergic reactions observed with enzymes obtainedfrom sporular strains.

We claim: 1. A process for selectingasporogenic bacteria from the genusBacillus subtilis which comprises:

a'pmutating germinated spores of wild Bacillus and spreading thesporessubjected to the mutagenic treatment over an agar containing nutrientbroth accommodated in a series of Petri dishes to obtain afterincubation colonies containing spores;

b. replicating each Petri dish of said series to obtain a firsthomologue series of Petri dishes;

c. submitting each Petri dish of said first mentioned series tochloroform vapor and replicating it to obtain a second homologue seriesof Petri dishes;

d. incubating said first homologue series and said second homologueseries of Petri dishes; I

e. by comparison between homologue Petri dishes of said first homologueseries and said second homologue series, selecting the asporogenicmutant strains which are strains of said Petri dishes of said firsthomologue series which are not in the Petri dishes of said secondhomologue series; and

f. testing proteolytic activities of these selected strains on casein.

2. Process for preparing protease without spore which comprisesculturing in a known manner for the wild Bacillus subtilis anasporogenie bacteria selected by the process of claim 1.

3. Process according to claim 2 wherein asporogenic bacteria is aBacillus subtilis of the type identified by No. 71-1-00] deposited inInstitut Pasteur de Paris.

2. Process for preparing protease without spore which comprisesculturing in a known manner for the wild Bacillus subtilis anasporogenic bacteria selected by the process of claim
 1. 3. Processaccording to claim 2 wherein asporogenic bacteria is a Bacillus subtilisof the type identified by No. 71-I-001 deposited in ''''Institut Pasteurde Paris.''''